The Roles And Underlying Mechanisms Of DNMT1and CLM-3in Innate Immune Response

DNA methyltransferases are highly conserved in prokaryotic and eukaryoticenzymes, they modified cytosine C5locus after the genome DNA replication, whichexert a variety of important physiological processes in the body, including: regulation ofgene expression, gene imprinting, maintain chromosomal integrity, and X-chromosomeinactivation. According to their different structure and function, mammalian DNAmethyltransferase (Dnmts) are divided into two categories: DNA methylationmaintenance enzyme Dnmt1and de novo DNA methyltransferase Dnmt3a,, Dnmt3b andDnmt3L.Dendritic cells (dendritic cells, DC) refers to a class of cells that process dendriticand stellate pleomorphic, high expression of MHC I and class I molecules, which caneffectively uptake, processing and handling of antigen to activate naive T cells. DC isfirst discovered by Steinman and Cohn in1973. As the most powerful professionalantigen-presenting cells (antigen presenting cell, APC), DC’s biggest feature is the abilityto stimulate naive T cells (naive T cell) activation and proliferation. DC is the trigger ofantigen-specific immune response, due to its unique position, the studies on DC providedeep insight into the generation and regulation mechanisms of immune response, cancer,transplant rejection, infection, autoimmune occurrence and development of diseases.which have important theoretical significance.Immunoglobulin superfamily (IgSF) consists of a large number of both membraneand intracellular receptors, which involve nearly every aspect of immune response bothin innate and adaptive immunity. The murine CMRF-35like molecule (CLM) family is apaired immunoreceptor family that belongs to the IgSF. Members of CLM familycontribute to both positive and negative regulation of immune responses uponrecognition of various ligands. We and others have identified several CLM receptors,such as DC-derived immunoglobulin receptor (DIgR)2/CLM-1/LMIR3/MAIR-V andDIgR1/CLM-4/LMIR2/MAIRII, which are preferentially expressed onantigen-presenting cells such as DCs and macrophages. We show that CLM-1, aninhibitory receptor, containing two ITIM motifs in the cytoplasmic region that associatedwith Src homology2-containting phosphatase-1(SHP-1), can negatively regulatesignaling of DC-initiated antigen-specific T-cell responses. Deficiency of CLM-1results in significantly enhanced nitric oxide and pro-inflammatory cytokine production inbone-marrow derived CD11c+cells, along with increased demyelination and worsenedclinical scores. To date, the roles of CLM members in the regulation of TLR signalingremain unclear.Antigen-presenting cells can detect the invading pathogens by recognizingpathogen-associated molecular patterns (PAMPs) through pattern recognition receptors(PRRs), and then trigger innate immune responses and prime antigen-specific adaptiveimmunity. Activation of innate immune cells can induce production of pro-inflammatorycytokines and type I interferons (IFNs), ultimately eliminating the invading pathogenicmicroorganisms. As one of most important kinds of PRRs, Toll-like receptors (TLRs) arehighly expressed in antigen-presenting cells including macrophages/monocytes, dendriticcells (DCs). According to the subcellular location of all TLR family members, TLRscould be classified into two categories: cell membrane-localized TLRs and intracellularTLRs. Among all the TLRs, the endosome-localized TLR9is the only one thatrecognizes pathogen-derived DNA. Once binding of bacterial CpG DNA or syntheticoligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-ODN), TLR9can directly activate antigen-presenting cells and innate immune cells to secret variouspro-inflammatory cytokines. Ligation of CpG-ODN to TLR9results in recruitment of theadaptor protein MyD88and the subsequent activation of downstream signaling pathwayswith the phosphorylation of MAPKs and NF-κB, ultimately leading to production ofpro-inflammatory cytokines and type I IFNs. It is believed that TLR9agonists couldinduce strong Th1immune response to show protective activity against bacterial andviral infection. However, how to selectively activate TLR9signaling so as to initiateeffective immune response and the underlying mechanisms for full activation ofTLR9-triggered innate response remain to be fully understood.Based on the recent research, we use modern experimental techniques to study therole of DNMT1in human monocyte-derived dendritic cells and mouse peritonealmacrophages during the immune response; and the function of CLM-3in the Toll-likereceptor signaling transduction during pathgen invading.Part I Identification of proteins that interacting with DNMT1inhuman monocyte-derived dendritic cellsWith the progress of studies on the role of mouse bone marrow-derived dendritic cells in the immune response, we began to focus on the function of humanmonocyte-derived dendritic cell. As a critical DNA methyltransferase, the functions ofDNMT1continue to be found, so we began to explore the role of DNMT1in humanmonocyte-derived dendritic cells.We use protein co-immunoprecipitation techniques, mass spectrometry compatiblesilver staining and LS-MS/MS, and detected a few proteins that interacting with DNMT1in human monocyte-derived dendritic cells, which are mainly concentrated on thetranslation initiation factor eIF family, including eIF3A, eIF3H; and cell cycle andapoptosis regulatory protein1(cell cycle and apoptosis regulator protein1, CARP1).As is reported, DNMT1mainly exerts the role of DNA methylation modification inthe nucleus, and eIF3A and eIF3H mainly iniate protein translation in the cytoplasm.These prompted us to explore whether DNMT1could play its non-classical features inthe cytoplasm and participated in several other biological activities. By usingco-immunoprecipitation, we found that DNMT1do exist in the cytoplasm, and interactedwith eIF3A and eIF3H, but we couldn’t detect any interaction between DNMT1andeIF3A or eIF3H in the nucleus. While the specifical role of DNMT1in the cytoplasmneeds further investigation.We also found that CARP1(cell cycle and apoptosis regulatory protein1) in the listthat maybe interact with DNMT1, after co-IP detection, we found that DNMT1couldmainly interact with CARP1in the nucleus, suggesting that CARP1might be involved inDNMT1classic features. CARP1could be a new molecule that participates in DNAmethylation, but its specific function needs further study.Part II The role of DNMT1in the innate response of macrophagesTLRs (Toll like receptors) are the important pattern recognition receptors, whichparticipate in the activation of innate immune response and regulation of adaptiveimmune responses against various pathogens. Due to insufficient activation of immuneresponses, the body couldn’t effectively remove invading pathogens, while duringexcessive immune activation would cause a series of injury, such as septic shock.Therefore, TLRs signaling pathway must be strictly regulated, and the cross-talk betweenTLR signaling pathway and other signaling pathways is one of the hotspots in recent years.Whether DNA methyltransferase DNMT1participate in the TLR signalingpathways is still unknown yet. After silencing the DNMT1in murine peritonealmacrophages, we stimulated the macrophages with a variety of TLR ligands, and thenQ-PCR examize the mRNA level of cytokines. Finally we found that DNMT1is unableto affect TLR3, TLR4and TLR9activation, with no obivious change of IFN-beta andTNF-alpha production.As previous reported, macrophages also plays an important role in the process ofanti-virus immune responses. We found that DNMT1interference can affect VSV andSeV induced IFN-beta production, DNMT1can promote the production of IFN-betaduring virus infection, suggesting that DNMT1might be involved in RIG-I signalingpathway, but its specific mechanism needs further research.Part III The molecular mechanism of CLM-3to regulate TLR9signaling in macrophagesCLM-3, a member of CLM family, is closely related to CLM-5, an activatingreceptor that transmits an activating signal by interacting with FcR among adaptormolecules containing ITAM or the related activating motif-bearing molecules. LikeCLM-5, CLM-3could associate with FcR; while the role of CLM-3in innate immunityhas not yet been identified. In the previous study, we demonstrated that CLM-3, which ispreferentially expressed in macrophages, is an endosome/lysosome locatedimmunoreceptor. CLM-3expression was downregulated in macrophages following thetreatment with TLR9ligand CpG-ODN. CLM-3can function as a positive regulator ofTLR9signaling in macrophages. In this study we showed that CLM-3can promoteTLR9signaling by augmenting the ubiquitination of TRAF6and subsequently enhancingdownstream MAPKs and NF-κB signaling, thus leading to the full activation ofTLR9-triggered innate immune response. Therefore, the findings suggest that crosstalkwith CLM-3immunoreceptor can promote TLR9-triggered innate immune responses,which may benefit host to eliminate the invading pathogens more potently.We observed the subcellular localization of CLM-3in Raw264.7cells, and foundthat CLM-3co-localized with lysosome-associated membrane protein1(LAMP1)-positive compartment and early endosome marker Rab5. No obvious co-localization with GM130, the representative marker of Gogi, was detected. Therefore,unlike other cell membrane-located CLMs, CLM-3was located in membrane-containingintracellular organelles endosome and lysosome.The subcellular localization of CLM-3in endosome and lysosome of macrophagesinspired us to investigate the roles of CLM-3in TLR-triggered activation ofmacrophages. We stimulated macrophages with TLR4,3,9ligands and found thatCLM-3expression was apparently down-regulated by CpG-ODN treatment. However,CLM-3expression remained almost unchanged following Poly I:C or LPS stimulation,indicating that CLM-3might be potentially involved in CpG-ODN/TLR9response.Furthermore, given to the observation that CLM-3resides in the endosome, weinvestigated whether overexpression of CLM-3could render the inhibitory effect ofchloroquine on the activation of TLR9pathway by CpG-ODN, but no reverse effect wasobserved in primary peritoneal macrophages from CLM-3-TG mice. Consistent with thatCLM-3expression was only regulated in response to CpG-ODN stimulation, these datasuggest that CLM-3may preferentially promote pro-inflammatory cytokine production inmacrophages triggered by TLR9but not TLR4or TLR3.The CLM-3transgenic mice (CLM-3-TG) were generated and used to observe therole of CLM-3in TLR9-triggered innate response. We identified that primary peritonealmacrophages derived from CLM-3-TG mice highly expressed HA tag-coupled CLM-3.In consistent with the in vitro observation that CLM-3overexpression could promoteCpG-ODN-induced TNF-α and IL-6expression, we found that the primary peritonealmacrophages derived from the CLM-3-TG mice could express higher level of TNF-α andIL-6, at both mRNA and protein level, than that from the littermate control mice inresponse to CpG-ODN stimulation. Coincidently, we observed more production ofTNF-α and IL-6in serum of CLM-3-TG mice after peritoneal injection ofCpG-ODN.These results further identified CLM-3as a positive regulator ofTLR9-triggered innate response.MAPKs and NF-κB signaling are key pathways for the CpG-ODN-initiatedcytokine production. Our previous studies showed that CLM-3enhancedCpG-ODN-induced activation of ERK1/2, JNK1/2and p38, and phosphorylation ofIKKα/β and IκBα in macrophages. In consistent with the observation, in this study we found that the enhanced CpG-ODN-induced activation of ERK1/2, JNK1/2and p38, andphosphorylation of IKKα/β and IκBα were observed in macropahges derived fromCLM-3-TG mice when stimulated with CpG-ODN. Consistently, more phosphorylatedp65was detected in the nucleus of primary peritoneal macrophages from CLM-3-TGmice after stimulated with CpG-ODN in vitro. Furthermore, NF-κB luciferase reporterassay showed that CLM-3overexpression could promote MyD88-mediated NF-κBactivity in HEK293cells. These results suggested that CLM-3promoted theCpG-ODN-initiated TLR9signaling pathway.After the engagement of TLR9by CpG-ODN, MyD88recruits the IL-1receptor-associated kinases IRAK1, IRAK2and IRAK4. Then IRAK activation recruitsTRAF6for downstream activation of the MAPKs and NF-κB. TRAF6is a key player inthe TLR9signaling pathway and ubiquitination of TRAF6is shown to be necessary foractivation of downstream signaling. So we wonder whether overexpression of CLM-3may enhance the ubiquitination of TRAF6. After silencing of CLM-3in primaryperitoneal macrophages, we detected the ubiquitination level of TRAF6in macrophagesonce stimulated with CpG-ODN, and found that silencing of CLM3reduced TRAF6ubiquitination. Coincidently, higher ubiquitination level of TRAF6in primary peritonealmacrophages from CLM-3-TG mice was observed once stimulated with CpG-ODN invitro. Then the subcellular location of CLM-3encouraged us to investigate whetherCLM-3could directly interact with TLR9, MyD88or TRAF6. We found CLM-3couldweakly interact with TLR9in primary peritoneal macrophages from CLM-3transgenicmice, but CLM-3could not interact with MyD88or TRAF6. Furthermore, the interactionof CLM-3and TLR9could be enhanced in macrophages after stimulation withCpG-ODN. These results suggested that CLM-3could promote the activation of TRAF6by promoting its ubiquitination through interaction with TLR9, ultimately enhancing thedownstream signaling pathways in response to CpG-ODN ligation.In summary, three parts of the above studies will be helpful to better understand themolecular regulation of the innate immune response. We found that DNMT1couldinteract with eIF3A, eIF3H, and CARP1, in human peripheral blood monocyte-deriveddendritic cells, although its potential role needs further study. In murine peritonealmacrophages, we observed that DNMT1could promote VSV and SeV induced IFN-betaproduction, indicating DNMT1may promote RIG-I-triggered innate response, so it’s thefirst time that DNMT1may be involved in the innate immune response, addition to its classic role as a DNA methyltransferase enzyme. By studying the role of CLM-3onTLR9signaling in macrophages, we found CLM-3could promote the ubiquitination ofTRAF6then enhance TLR9CpG-ODN induced immune responses. and upon stimulationof CpG-ODN, the binding between CLM-3and TLR9could be enhanced, thus providingthe mechanistic explanation for our previous observation that CLM-3could enhance thephosphorylation of MAPKs and NF-κB after CpG-ODN activation. The study onDNMT1and CLM-3broadens our view of innate immune recognition, which is alsoexpected to be applied in seeking treatment methods on infection immunity and cancer.